Pediatric renal tumors account for about six percent of all childhood cancers in the United States. (1) The survival rate of pediatric patients with kidney tumors varies according to the respective pathology. Wilms"" tumor has a good prognosis, with about an 80% cure rate; Clear Cell Sarcoma (CCS) of the kidney has a fair prognosis with about a 60% cure rate; whereas Rhabdoid tumors (RTK) of the kidney has a poor prognosis, with a cure rate of only about 10%. Despite this association, it is not uncommon to find that the behavior of a specific tumor is disparate with its pathologic diagnosis. Unfortunately, there are no recognized biologic markers (except for clinical stage and tumor type) for predicting prognosis or for guiding treatment, follow-up and counseling of pediatric patients with renal tumors.
Apoptosis, or programmed cell death, is the process by which activated cells undergo a suicide program that results in individual cell death. (2) It is a highly orchestrated process where cells die in a regulated fashion. Aberration in the process of apoptosis may contribute to the pathogenesis of certain tumors. (3) It has been shown that pro-apoptotic receptors were expressed at greater levels in Wilms"" tumors of good prognosis, compared to CCS or RTK. (4) Surprisingly, no association was found with anti-apoptotic factors like Bcl-2 or Bcl-XL and prognosis.
An excess of apoptosis inhibitors could protect dividing cells from dying, and thus contribute to tumorigenicity. A related family of Inhibitors of Apoptosis proteins (IAP) has been described in many species and these proteins block apoptosis by direct inhibition of downstream effector caspases in the pathway of cell death. (5,6) A novel member of the IAP gene family designated Survivin was recently described and shown to be associated with tumors of poor prognosis. (7-10) Survivin is selectively expressed in tissues during development but not in terminally differentiated mature tissue and is also expressed in several pediatric renal tumors tested. (11) Survivin appears to block default induction of apoptosis during mitosis, and when over expressed in cancer cells, Survivin may permit aberrant proliferation through mitosis.
Currently, for many types of tumors there are no available markers specific for the tumor type. Thus, for these tumor types, treatment cannot be specifically guided nor can prognosis be predicted. Therefore, there is an ongoing need for methods and kits to predict recurrent tumor growth, in particular solid tumors and hematologic cancers. Such an indicator would be helpful not only for guiding the patient as to outcome, but also for selecting treatment groups as well as follow-up during treatment of disease.
The present invention provides a diagnostic method for predicting the recurrence of a tumor or cancer in a mammal by contacting RNA from a mammalian physiological sample suspected of being tumorigenic or cancerous with a Survivin-specific oligonucleotide comprising a first label, and a pro-apoptosis factor (PAF)-specific oligonucleotide comprising a second label under conditions effective to hybridize the RNA to the oligonucleotides so as to yield a first population of RNA labeled with the Survivin-specific oligonucleotide and a second population of RNA labeled with the PAF-specific oligonucleotide; quantifying the first and second populations of labeled RNA to determine an amount of Survivin RNA and an amount of PAF RNA present in the sample; and calculating the ratio of the amount of Survivin RNA and the amount of PAF RNA; wherein a Survivin:PAF ratio of more than about 1.5 is predictive that the tumor will recur. The Survivin:PAF ratio may be more than about 1.6, or even more than about 2.0. The PAF may be Fas, BID, p53, DR4 (TRAIL-R1), DR5 (TRAIL-R2), or TNF-R. The physiological sample may be a tissue sample, such as a tissue-lysate protein sample. The tissue may be any solid tumor, encompassing childhood and adult tumors. Pediatric examples include Neuroblastoma, Pediatric renal tumors, Hepatoblastoma, Rhabdomysosarcoma and undifferentiated sarcomas as well as germ cell and endocrine tumors. Adult examples would include tumors of the nervous system, gastrointestinal and urogenital tract, as well as all types of sarcomas. Alternatively, the physiological sample may be a fluid, such as whole blood or blood serum.
The present invention also provides a diagnostic method for predicting the recurrence of a tumor or cancer in a mammal by contacting a mammalian tissue sample suspected of being tumorigenic or cancerous with a Survivin-specific ligand comprising a first label, and a pro-apoptosis factor (PAF)-specific ligand comprising a second label under conditions effective to hybridize protein present in the tissue sample to the ligands so as to yield a first population of protein hybridized to the Survivin-specific ligand and a second population of protein hybridized to the PAF-specific ligand; quantifying the first and second populations of labeled protein to determine an amount of Survivin and an amount of PAF present in the sample; and calculating the ratio of the amount of Survivin and the amount of PAF; wherein a Survivin:PAF ratio of more than about 1.5 is predictive that the tumor will recur. The Survivin:PAF ratio may be more than about 1.6, or even more than about 2.0. The PAF may be Fas, BID, p53, DR4 (TRAIL-R1), DR5 (TRAIL-R2), or TNF-R. The physiological sample may be a tissue sample, such as a tissue-lysate protein sample. The tissue may be any solid tumor, encompassing childhood and adult tumors. Pediatric examples include Neuroblastoma, Pediatric renal tumors, Hepatoblastoma, Rhabdomysosarcoma and undifferentiated sarcomas as well as germ cell and endocrine tumors. Adult examples would include tumors of the nervous system, gastrointestinal and urogenital tract, as well as all types of sarcomas. Alternatively, the physiological sample may be a fluid, such as whole blood or blood serum. The agent may be an antibody, in particular a population of polyclonal or monoclonal antibodies.
The present invention further provides a diagnostic kit for predicting recurrence of tumor or cancer in a mammal, containing packaging material, a Survivin-specific ligand, a PAF-specific ligand and instructions means directing the use of (a) and (b) in accord with the above described methods.
FIG. 1: Methods of determining quantity of pro- and anti-apoptotic factors present in a tissue sample.
FIG. 2: Western blot analysis of several pediatric renal tumors and corresponding RPA values. NK=Normal Kidney; WTII=Wilms"" tumor stage II; RTK=Rhabdoid tumor of the kidney; CCS=Clear cell sarcoma of the kidney. RPA=RNase protection assay calculated as percent of L32 expression.
FIG. 3: Autoradiographic plate of RNase protection assay (RPA) showing bands marking the various mRNA species detected in four examples of pediatric renal tumors studied. WTI=Wilms"" tumor stage I, RTK IIIP=Rhabdoid tumor stage III, CCS III and IV=Clear cell sarcoma stage III and IV.
FIG. 4: Comparative chart for the Survivin:Fas ration calculated from RPA values for normal kidney, non-recurrent tumors, and recurrent tumors.
FIG. 5: Survivin immunoreactivity in examples of histologic specimens. A=Normal kidney, B=RTK Negative control (absent primary Ab) and C=RTK (Rhabdoid tumor of the kidney). Note positive (dark brown) intra-cellular Survivin staining in plate C and absence of Survivin staining in A and B.
FIG. 6: The amount of TNF related, Bcl-2 related, and Survivin related RNA present in tumors as quantified radio-analytically (as measured as a percent of L32).
FIG. 7: Pediatric Renal Tumors. TNF related receptors (i.e. Fas, DR5 and TNF-R) that are differentially expressed in pediatric renal tumors.